Glucosylation Mediated Rolling Circle Amplification Combined with a qPCR Assay for the Detection of 5-Hydroxymethylcytosine.

Abstract

The detection of 5-hydroxymethylcytosine (5hmC), a newly recognized epigenetic mark, is essential to its functional study. Here, an efficient and simple two-step-amplification method to detect 5hmC mediated by glucosylation is reported, which combines rolling circle amplification (RCA) and a quantitative polymerase chain reaction (qPCR). In the first step RCA, the glucosylated 5hmC (5ghmC), but not 5hmC, 5-methylcytosine (5mC) or cytosine (C) bases, could directly and specifically inhibit the activity of phi29 DNA polymerase, resulting in less RCA product compared to that using C-/5mC-/5hmC-containing templates. Then, the second step qPCR is adopted to test and verify the difference of the product quantity of 5ghmC-related RCA. The results show that the delta cycle threshold, ΔCt, obtained by subtracting the cycle threshold value (Ct) of C-related qPCR from that of each qPCR, of 5ghmC-related qPCR reaches 1.59 ± 0.03, significantly different from that of C-/5mC-/5hmC-related qPCR (-0.00 ± 0.09, 0.06 ± 0.08 and -0.02 ± 0.03, respectively). Meanwhile, a linear relationship is observed between the 5ghmC levels and the ΔCt values. This suggests that the strategy has a potential application for 5hmC detection and quantification.